Bottom Line:
Rubella virus (RuV) infection of pregnant women can cause fetal death, miscarriage, or severe fetal malformations, and remains a significant health problem in much of the underdeveloped world.Other tested cations did not substitute.Alanine substitution of N88 or D136 was lethal.

Affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACTRubella virus (RuV) infection of pregnant women can cause fetal death, miscarriage, or severe fetal malformations, and remains a significant health problem in much of the underdeveloped world. RuV is a small enveloped RNA virus that infects target cells by receptor-mediated endocytosis and low pH-dependent membrane fusion. The structure of the RuV E1 fusion protein was recently solved in its postfusion conformation. RuV E1 is a member of the class II fusion proteins and is structurally related to the alphavirus and flavivirus fusion proteins. Unlike the other known class II fusion proteins, however, RuV E1 contains two fusion loops, with a metal ion complexed between them by the polar residues N88 and D136. Here we demonstrated that RuV infection specifically requires Ca(2+) during virus entry. Other tested cations did not substitute. Ca(2+) was not required for virus binding to cell surface receptors, endocytic uptake, or formation of the low pH-dependent E1 homotrimer. However, Ca(2+) was required for low pH-triggered E1 liposome insertion, virus fusion and infection. Alanine substitution of N88 or D136 was lethal. While the mutant viruses were efficiently assembled and endocytosed by host cells, E1-membrane insertion and fusion were specifically blocked. Together our data indicate that RuV E1 is the first example of a Ca(2+)-dependent viral fusion protein and has a unique membrane interaction mechanism.

ppat-1004530-g005: Impact of Ca2+ on RuV E1 low-pH triggered conformational change.(A) Low pH inactivation of RuV infectivity. RuV was adsorbed to poly-D-lysine coated wells and incubated for 15 min at 37°C in fusion media of the indicated pH and containing 2 mM CaCl2 or 1.5 mM EDTA where indicated. The wells were then washed, overlaid with Vero cells, incubated for 1 h at 37°C in complete medium with calcium to permit virus entry, and cultured for 48 h in presence of NH4Cl. Nuclei were stained with Hoescht and infection was quantitated by immunofluorescence. (B) Quantitation of data from (A). Infectivity was calculated relative to the control treated with buffer at pH 7.0. Error bars represent the standard deviation from the mean of 3 independent experiments. (C–D) E1 trypsin-resistance assay. (C) Purified radiolabeled RuV was treated at the indicated pH for 5 min at 37°C in the presence of 2 mM CaCl2 or 1.5 mM EDTA as indicated. Samples were digested with trypsin with or without inhibitors, and the E1 protein analyzed by immunoprecipitation and SDS-PAGE. (D) Quantitation of E1 trypsin resistance assays performed as in (C). Graph shows the mean and standard deviation of 3 independent experiments.

Mentions:
Low pH triggers the irreversible rearrangement of the prefusion RuV E2-E1 heterodimer to the E1 hairpin-like homotrimer, driving fusion of the virus with a target membrane [24], [28] or virus inactivation in the absence of target membranes [24]. To determine if the role of Ca2+ in fusion is via effects on this conformational change, we first examined the effect of Ca2+ on low pH inactivation [36]. RuV particles were immobilized on poly-D-lysine-coated wells, treated with neutral or low pH buffers in the presence or absence of CaCl2 or EDTA, overlaid with Vero cells and incubated for 48 h in growth media to quantitate virus infectivity (Fig. 5A and B). Fluorescence microscopy confirmed that buffer treatments did not cause elution of the adsorbed virus from the wells (Fig. 5A, Fig. S4). Virus infectivity was maintained after treatment at neutral pH whether or not Ca2+ or EDTA was present, but low pH caused virus inactivation under either condition.

ppat-1004530-g005: Impact of Ca2+ on RuV E1 low-pH triggered conformational change.(A) Low pH inactivation of RuV infectivity. RuV was adsorbed to poly-D-lysine coated wells and incubated for 15 min at 37°C in fusion media of the indicated pH and containing 2 mM CaCl2 or 1.5 mM EDTA where indicated. The wells were then washed, overlaid with Vero cells, incubated for 1 h at 37°C in complete medium with calcium to permit virus entry, and cultured for 48 h in presence of NH4Cl. Nuclei were stained with Hoescht and infection was quantitated by immunofluorescence. (B) Quantitation of data from (A). Infectivity was calculated relative to the control treated with buffer at pH 7.0. Error bars represent the standard deviation from the mean of 3 independent experiments. (C–D) E1 trypsin-resistance assay. (C) Purified radiolabeled RuV was treated at the indicated pH for 5 min at 37°C in the presence of 2 mM CaCl2 or 1.5 mM EDTA as indicated. Samples were digested with trypsin with or without inhibitors, and the E1 protein analyzed by immunoprecipitation and SDS-PAGE. (D) Quantitation of E1 trypsin resistance assays performed as in (C). Graph shows the mean and standard deviation of 3 independent experiments.

Mentions:
Low pH triggers the irreversible rearrangement of the prefusion RuV E2-E1 heterodimer to the E1 hairpin-like homotrimer, driving fusion of the virus with a target membrane [24], [28] or virus inactivation in the absence of target membranes [24]. To determine if the role of Ca2+ in fusion is via effects on this conformational change, we first examined the effect of Ca2+ on low pH inactivation [36]. RuV particles were immobilized on poly-D-lysine-coated wells, treated with neutral or low pH buffers in the presence or absence of CaCl2 or EDTA, overlaid with Vero cells and incubated for 48 h in growth media to quantitate virus infectivity (Fig. 5A and B). Fluorescence microscopy confirmed that buffer treatments did not cause elution of the adsorbed virus from the wells (Fig. 5A, Fig. S4). Virus infectivity was maintained after treatment at neutral pH whether or not Ca2+ or EDTA was present, but low pH caused virus inactivation under either condition.

Bottom Line:
Rubella virus (RuV) infection of pregnant women can cause fetal death, miscarriage, or severe fetal malformations, and remains a significant health problem in much of the underdeveloped world.Other tested cations did not substitute.Alanine substitution of N88 or D136 was lethal.

Affiliation:
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACTRubella virus (RuV) infection of pregnant women can cause fetal death, miscarriage, or severe fetal malformations, and remains a significant health problem in much of the underdeveloped world. RuV is a small enveloped RNA virus that infects target cells by receptor-mediated endocytosis and low pH-dependent membrane fusion. The structure of the RuV E1 fusion protein was recently solved in its postfusion conformation. RuV E1 is a member of the class II fusion proteins and is structurally related to the alphavirus and flavivirus fusion proteins. Unlike the other known class II fusion proteins, however, RuV E1 contains two fusion loops, with a metal ion complexed between them by the polar residues N88 and D136. Here we demonstrated that RuV infection specifically requires Ca(2+) during virus entry. Other tested cations did not substitute. Ca(2+) was not required for virus binding to cell surface receptors, endocytic uptake, or formation of the low pH-dependent E1 homotrimer. However, Ca(2+) was required for low pH-triggered E1 liposome insertion, virus fusion and infection. Alanine substitution of N88 or D136 was lethal. While the mutant viruses were efficiently assembled and endocytosed by host cells, E1-membrane insertion and fusion were specifically blocked. Together our data indicate that RuV E1 is the first example of a Ca(2+)-dependent viral fusion protein and has a unique membrane interaction mechanism.